Design of vectors for efficient expression of human purine nucleoside phosphorylase in skin fibroblasts from enzyme-deficient humans.
Design of vectors for efficient expression of human purine nucleoside phosphorylase in skin fibroblasts from enzyme-deficient humans.
复制标题
设计用于在酶缺陷人类的皮肤成纤维细胞中有效表达人嘌呤核苷磷酸化酶的载体。
DOI：
10.1073/pnas.85.18.6851
复制
发表时间：
1988
影响因子：
11.1
通讯作者：
Miller,AD
中科院分区：
文献类型：
--
作者：
Osborne,WR;Miller,AD
嘌呤核苷磷酸化酶(PNP)缺乏症是一种遗传性疾病，与严重的免疫缺陷相关，是一种致命性疾病。酶替代疗法是治疗这种疾病的一种有吸引力的方法。为此，我们构建了含有人PNP基因和编码新霉素磷酸转移酶的可选基因的逆转录病毒载体。PNP的表达受猴病毒40早期启动子、人巨细胞病毒直接早期启动子或逆转录病毒启动子控制。来自两名感染这些载体的无关PNP缺陷患者的培养皮肤成纤维细胞表达的平均PNP活性分别为每毫克蛋白0.03、0.74和5.9摩尔/小时。后者的PNP活性是正常皮肤成纤维细胞中观察到的每毫克蛋白质0.74摩尔/小时水平的8倍，使外源脱氧鸟苷能够快速代谢，外源脱氧鸟苷是积累在PNP缺陷患者血浆中的细胞毒性代谢物。这些实验表明，病毒长末端重复序列是PNP表达的最强启动子，并暗示了人皮肤成纤维细胞作为治疗性基因表达载体的潜力。
Purine nucleoside phosphorylase (PNP; purine-nucleoside orthophosphate ribosyltransferase, EC 2.4.2.1) deficiency is an inherited disorder associated with a severe immune defect that is fatal. Enzyme replacement therapy is an attractive approach to treatment of this disease. To this aim we constructed retroviral vectors containing a human PNP cDNA and a selectable gene encoding neomycin phosphotransferase. PNP expression was controlled by either the early promoter from simian virus 40, the immediate early promoter from human cytomegalovirus, or the retroviral promoter. Cultured skin fibroblasts from two unrelated PNP-deficient patients that were infected with these vectors expressed mean PNP activities of 0.03, 0.74, and 5.9 mumol/hr per mg of protein, respectively. The latter infectants had PNP activities eight times the level of 0.74 mumol/hr per mg of protein observed in normal skin fibroblasts, enabling rapid metabolism of exogenous deoxyguanosine, the cytotoxic metabolite that accumulates in the plasma of PNP-deficient patients. These experiments indicate that viral long terminal repeat was the strongest promoter for expression of PNP and suggest the potential of human skin fibroblasts as vehicles for therapeutic gene expression.